Papilloma virus capsomere vaccine formulations and method of use

ABSTRACT

Vaccine formulations comprising viral capsomeres are disclosed along with methods for their production. Therapeutic and prophylactic methods of use for the vaccine formulations are also disclosed.

FIELD OF THE INVENTION

[0001] The present invention relates to vaccine formulations comprisingpapilloma virus proteins, either as fusion proteins, truncated proteins,or truncated fusion proteins. The invention further embraces methods forproducing capsomeres of the formulations, as well as prophylactic andtherapeutic methods for their use.

BACKGROUND

[0002] Infections with certain high-risk strains of genital papillomaviruses in humans (HPV)—for example, HPV 16, 18, or 45—are believed tobe the main risk factor for the formation of malignant tumors of theanogenital tract. Of the possible malignancies, cervical carcinoma is byfar the most frequent: according to an estimate by the World HealthOrganization (WHO), almost 50,000 new cases of the disease occurannually. Because of the frequency with which this pathology occurs, theconnection between HPV infection and cervical carcinoma has beenextensively examined, leading to numerous generalizations.

[0003] For example, precursor lesions of cervical intraepithelialneoplasia (CIN) are known to be caused by papilloma virus infections[Crum, New Eng. J. Med. 310:880-883 (1984)]. DNA from the genomes ofcertain HPV types, including for example, strains 16, 18, 33, 35, and45, have been detected in more than 95% of tumor biopsies from patientswith this disorder, as well as in primary cell lines cultured from thetumors. Approximately 50 to 70% of the biopsied CIN tumor cells havebeen found to include DNA derived only from HPV 16.

[0004] The protein products of the HPV 16 and HPV 18 early genes E6 andE7 have been detected in cervical carcinoma cell lines as well as inhuman keratinocytes transformed in vitro [Wettstein, et al., inPAPILLOMA VIRUSES AND HUMAN CANCER, Pfister (Ed.), CRC Press: BocaRaton, Fla. 1990 pp 155-179] and a significant percentage of patientswith cervical carcinoma have anti-E6 or anti-E7 antibodies. The E6 andE7 proteins have been shown to participate in induction of cellular DNAsynthesis in human cells, transformation of human keratinocytes andother cell types, and tumor formation in transgenic mice [Arbelt, etal., J. Virol., 68:4358-4364 (1994); Auewarakul, et al., Mol. Cell.Biol. 14:8250-8258 (1994); Barbosa. et al., J. Virol. 65:292-298 (1991);Kaur, et al, J. Gen. Virol. 70:1261-1266 (1989); Schlegel, et al., EMBOJ., 7:3181-3187 (1988)]. The constitutive expression of the E6/E7proteins appears to be necessary to maintain the transformed conditionof HPV-positive tumors.

[0005] Despite the capacity of some HPV strains to induce neoplasticphenotypes in vivo and in vitro, still other HPV types cause benigngenital warts such as condylomata acuminata and are only rarelyassociated with malignant tumors [Ikenberg, In Gross, et al., (eds.)GENITAL PAPILLOMAVIRUS INFECTIONS, Springer Verlag: Berlin, pp.,87-112]. Low risk strains of this type include, for example, HPV 6 and11.

[0006] Most often, genital papilloma viruses are transmitted betweenhumans during intercourse which in many instances leads to persistentinfection in the anogenital mucous membrane. While this observationsuggests that either the primary infection induces an inadequate immuneresponse or that the virus has developed the ability to avoid immunesurveillance, other observations suggest that the immune system isactive during primary manifestation as well as during malignantprogression of papilloma virus infections [Altmann et al. in VIRUSES ANDCANCER, Minson et al., (eds.) Cambridge University Press, (1994) pp.71-80].

[0007] For example, the clinical manifestation of primary infection byrabbit and bovine papilloma virus can be prevented by vaccination withwart extracts or viral structural proteins [Altmann, et al., supra;Campo, Curr. Top. In Microbiol and Immunol. 186:255-266 (1994); Yindleand Frazer, Curr. Top. In Microbiol. and Immunol. 186;217-253 (1994)].Rodents previously vaccinated with vaccinia recombinants encoding HPV 16early proteins E6 or E7, or with synthetic E6 or B7 peptides, aresimilarly protected from tumor formation after inoculation of HPV 16transformed autologous cells [Altman, et al., supra; Campo, et al.,supra; Yindle and Frazer, et al. supra]. Regression of warts can beinduced by the transfer of lymphocytes from regressor animals followinginfection by animal papilloma viruses. Finally, in immunosuppressedpatients, such as, for example, recipients of organ transplants orindividuals infected with HIV, the incidence of genital warts. CIN, andanogenital cancer is elevated.

[0008] To date, no HPV vaccinations have been described which comprisehuman papilloma virus late L1 protein in the form of capsomeres whichare suitable both for prophylactic and therapeutic purposes. Since theL1 protein is not present in malignant genital lesions, vaccination withL1 protein does not have any therapeutic potential for these patients.Construction of chimeric proteins, comprising amino acid residues fromL1 protein and, for example E6 or E7 protein, which give rise tochimeric capsomeres, combines prophylactic and therapeutic functions ofa vaccine. A method for high level production of chimeric capsomereswould therefore be particularly desirable, in view of the possibleadvantages offered by such a vaccine for prophylactic and therapeuticintervention.

[0009] Thus there exists a need in the art to provide vaccineformulations which can prevent or treat HPV infection. Methods toproduce vaccine formulations which overcome problems known in the art tobe associated with recombinant HPV protein expression and purificationwould manifestly be useful to treat the population of individualsalready infected with HPV as well as useful to immunize the populationof individuals susceptible to HPV infection.

SUMMARY OF THE INVENTION

[0010] The present invention provides therapeutic and prophylacticvaccine formulations comprising chimeric human papilloma capsomeres. Theinvention also provides therapeutic methods for treating patientsinfected with an HPV as well as prophylactic methods for preventing HPVinfection in a susceptible individual. Methods for production andpurification of capsomeres and proteins of the invention are alsocontemplated.

[0011] In one aspect of the invention, prophylactic vaccinations forprevention of HPV infection are considered which incorporate thestructural proteins L1 and L2 of the papilloma virus. Development of avaccine of this type faces significant obstacles because papillomaviruses cannot be propagated to adequate titers in cell cultures orother experimental systems to provide the viral proteins in sufficientquantity for economical vaccine production. Moreover, recombinantmethodologies to express the proteins are not always straightforward andoften results in low protein yield. Recently, virus-like particles(VLPs), similar in make up to viral capsid structures, have beendescribed which are formed in Sf-9 insect cells upon expression of theviral proteins L1 and L2 (or L1 on its own) using recombinant vacciniaor baculovirus. Purification of the VLPs can be achieved very simply bymeans of centrifugation in CsCl or sucrose gradients [Kimbauer, et al.,Proc. Natl. Acad. Sci. (USA), 99:12180-12814 (1992): Kirnbaurer, et al.,J. Virol. 67:6929-6936 (1994); Proso, et al., J. Virol. 6714:1936-1944(1992): Sasagawa, et al., Virology 2016:126-195 (1995): Volpers, et al.,J. Virol. 69:3258-3264 (1995); Zhou, et al., J. Gen. Virol. 74:762-769(1993): Zhou, et al., Virology 185:251-257 (1991)]. WO 93/02184describes a method in which papilloma virus-like particles (VLPs) areused for diagnostic applications or as a vaccine against infectionscaused by the papilloma virus. WO 94/00152 describes recombinantproduction of L1 protein which mimics the conformational neutralizingepitope on human and animal papilloma virions.

[0012] In another aspect of the invention, therapeutic vaccinations areprovided to relieve complications of, for example, cervical carcinoma orprecursor lesions resulting from papilloma virus infection, and thusrepresent an alternative to prophylactic intervention. Vaccinations ofthis type may comprise early papilloma virus proteins, principally E6 orE7, which are expressed in the persistently infected cells. It isassumed that, following administration of a vaccination of this type,cytotoxic T-cells might be activated against persistently infected cellsin genital lesions. The target population for therapeutic interventionis patients with HPV-associated pre-malignant or malignant genitallesions. PCT patent application WO 93/20844 discloses that the earlyprotein E7 and antigenic fragments thereof of the papilloma virus fromHPV or BPV is therapeutically effective in the regression, but not inthe prevention, of papilloma virus tumors in mammals. While early HPVproteins have been produced by recombinant expression in E. coli orsuitable eukaryotic cell types, purification of the recombinant proteinshas proven difficult due to inherent low solubility and complexpurification procedures which generally require a combination of steps,including ion exchange chromatography, gel filtration and affinitychromatography.

[0013] According to the present invention, vaccine formulationscomprising papilloma virus capsomeres are provided which compriseeither: (i) a first protein that is an intact viral protein expressed asa fusion protein comprised in part of amino acid residues from a secondprotein; (ii) a truncated viral protein: (iii) a truncated viral proteinexpressed as a fusion protein comprised in part of amino acid residuesfrom a second protein, or (iv) some combination of the three types ofproteins. According to the invention, vaccine formulations are providedcomprising capsomeres of bovine papilloma virus (BPV) and humanpapilloma virus. Preferred bovine virus capsomeres comprise protein frombovine papilloma virus type I. Preferred human virus capsomeres compriseproteins from any one of human papilloma virus strains HPV6, HPV 11,HPV16, HPV18, HPV33, HPV35, and HPV45. The most preferred vaccineformulations comprise capsomeres comprising proteins from HPV16.

[0014] In one aspect, capsomere vaccine formulations of the inventioncomprise a first intact viral protein expressed as a fusion protein withadditional amino acid residues from a second protein. Preferred intactviral proteins are the structural papilloma vial proteins L1 and L2.Capsomeres comprised of intact viral protein fusions may be producedusing the L1 and L2 proteins together or the L1 protein alone. Preferredcapsomeres are made up entirely of L1 fusion proteins, the amino acidsequence of which is set out in SEQ ID NO: 2 and encoded by thepolynucleotide sequence of SEQ ID NO: 1. Amino acids of the secondprotein can be derived from numerous sources (including amino acidresidues from the first protein) as long as the addition of the secondprotein amino acid residues to the first protein permits formation ofcapsomeres. Preferably, addition of the second protein amino acidresidues inhibits the ability of the intact viral protein to formvirus-like particle structures; most preferably, the second proteinamino acid residues promote capsomere formation. In one embodiment ofthe invention, the second protein may be any human tumor antigen, viralantigen, or bacterial antigen which is important in stimulating animmune response in neoplastic or infectious disease states. In apreferred embodiment, the second protein is also a papilloma virusprotein. It also preferred that the second protein be the expressionproduct of papilloma virus early gene. It is also preferred, however,that the second protein be selected from group of E1, E2, E3, E4, E5,E6, and E7—early gene products encoded in the genome of papilloma virusstrains HVP6. HPV11, HPV18, HPV33, HPV35, or HPV 45. It is mostpreferred that the second protein be encoded by the HPV16 E7 gene, theopen reading frame of which is set out in SEQ ED NO: 3. Capsomeresassembled from fusion protein subunits are referred to herein aschimeric capsomeres. In one embodiment, the vaccine formulation of theinvention is comprised of chimeric capsomeres wherein L1 protein aminoacid residues make up approximately 50 to 99% of the total fusionprotein amino acid residues. In another embodiment, L1 amino acidresidues make up approximately 60 to 90% of the total fusion proteinamino acid residues; in a particularly preferred embodiment, L1 aminoacids comprise approximately 80% of the fusion protein amino acidresidues.

[0015] In another aspect of the invention, capsomere vaccineformulations are provided that are comprised of truncated viral proteinshaving a deletion of one or more amino acid residues necessary forformation of a virus-like particle. It is preferred that the amino aciddeletion not inhibit formation of capsomeres by the truncated protein,and it is most preferred that the deletion favor capsomere formation.Preferred vaccine formulations of this type include capsomeres comprisedof truncated L1 with or without L2 viral proteins. Particularlypreferred capsomeres are comprised of truncated L1 proteins. Truncatedproteins contemplated by the invention include those having one or moreamino acid residues deleted from the carboxy terminus of the protein, orone or more amino acid residues deleted from the amino terminus of theprotein, or one or more amino acid residues deleted from an internalregion (i.e., not from either terminus) of the protein. Preferredcapsomere vaccine formulations are comprised of proteins truncated atthe carboxy terminus. In formulations including L1 protein derived fromHPV16, it is preferred that from 1 to 34 carboxy terminal amino acidresidues are deleted. Relatively shorter deletions are also contemplatedwhich offer the advantage of minor modification of the antigenicproperties of the L1 proteins and the capsomeres formed thereof. It ismost preferred, however, that 34 amino acid residues be deleted from theL1 sequence, corresponding to amino acids 472 to 505 in HPV16 set out inSEQ ID NO: 2, and encoded by the polynucleotide sequence correspondingto nucleotides 1414 to 1516 in the human HPV16 L1 coding sequence setout in SEQ ID NO: 1.

[0016] When a capsomere vaccine formulation is made up of proteinsbearing an internal deletion, it is preferred that the deleted aminoacid sequence comprise the nuclear localization region of the protein.In the L1 protein of HPV 16, the nuclear localization signal is foundfrom about amino acid residue 499 to about amino acid residue 505.Following expression of L1 proteins wherein the NLS has been deleted,assembly of capsomere structures occurs in the cytoplasm of the hostcell. Consequently, purification of the capsomeres is possible from thecytoplasm instead of from the nucleus where intact L1 proteins assembleinto capsomeres. Capsomeres which result from assembly of truncatedproteins wherein additional amino acid sequences do not replace thedeleted protein sequences are necessarily not chimeric in nature.

[0017] In still another aspect of the invention, capsomere vaccineformulations are provided comprising truncated viral protein expressedas a fusion protein adjacent amino acid residues from a second protein.Preferred truncated viral proteins of the invention are the structuralpapilloma viral proteins L1 and L2. Capsomeres comprised of truncatedviral protein fusions may he produced using L1 and L2 protein componentstogether or L1 protein alone. Preferred capsomeres are those comprisedof L1 protein amino acid residues. Truncated viral protein components ofthe fusion proteins include those having one or more amino acid residuesdeleted from the carboxy terminus of the protein, or one or more aminoacid residues deleted from the amino terminus of the protein, or one ormore amino acid residues deleted from an internal region (i.e., not fromeither terminus) of the protein. Preferred capsomere vaccineformulations are comprised of proteins truncated at the carboxyterminus. In those formulations including L1 protein derived from HPV16,it is preferred that from 1 to 34 carboxy terminal amino acid residuesare deleted. Relatively shorter deletions are also contemplated thatoffer the advantage of minor modification of the antigenic properties ofthe L1 protein component of the fusion protein and the capsomeres formedthereof. It is most preferred, however, that 34 amino acid residues bedeleted from the L1 sequence, corresponding to amino acids 472 to 505 inHPV16 set out in SEQ ID NO: 2, and encoded by the polynucleotidesequence corresponding to nucleotides 1414 to 1516 in the human HPV16 L1coding sequence set out in SEQ ID NO: 1. When the vaccine formulation iscomprised of capsomeres made up of proteins bearing an internaldeletion, it is preferred that the deleted amino acid sequence comprisethe nuclear localization region, or sequence, of the protein.

[0018] Amino acids of the second protein can be derived from numeroussources as long as the addition of the second protein amino acidresidues to the first protein permits formation of capsomeres.Preferably, addition of the second protein amino acid residues promotesor favors capsomere formation. Amino acid residues of the second proteincan be derived from numerous sources, including amino acid residues fromthe first protein. In a preferred embodiment, the second protein is alsoa papilloma virus protein. It also preferred that the second protein bethe expression product of papilloma virus early gene. It is mostpreferred, however, that the second protein be selected from group ofearly gene products encoding by papilloma virus E1, E2, B, E4, E5, E6,and E7 genes. In one embodiment, the vaccine formulation of theinvention is comprised of chimeric capsomeres wherein L1 protein aminoacid residues make up approximately 50 to 99% of the total fusionprotein amino acid residues. In another embodiment, L1 amino acidresidues make up approximately 60 to 90% of the total fusion proteinamino acid residues; in a particularly preferred embodiment, L1 aminoacids comprise approximately 80% of the fission protein amino acidresidues.

[0019] In a preferred embodiment of the invention, proteins of thevaccine formulations are produced by recombinant methodologies, but informulations comprising intact viral protein, the proteins may beisolated from natural sources. Intact proteins isolated from naturalsources may be modified in vitro to include additional amino acidresidues to provide a fusion protein of the invention using covalentmodification techniques well known and routinely practiced in the art.Similarly, in formulations comprising truncated viral proteins theproteins may be isolated from natural sources as intact proteins andhydrolyzed in vitro using chemical hydrolysis or enzymatic digestionwith any of a number of site-specific or general proteases, thetruncated protein subsequently modified to include additional amino acidresides as described above to provide a truncated fusion protein of theinvention.

[0020] In producing capsomeres, recombinant molecular biology techniquescan be utilized to produce DNA encoding either the desired intactprotein, the truncated protein, or the truncated fusion protein.Recombinant methodologies required to produce a DNA encoding a desiredprotein are well known and routinely practiced in the art. Laboratorymanuals, for example Sambrook, et al., (eds.), MOLECULAR CLONING: ALABORATORY MANUAL. Cold Spring Harbor Press: Cold Spring Harbor, N.Y.(1989) and Ausebel et al., (eds.). PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons. Inc. (1994-1997), describe in detail techniques necessaryto carry out the required DNA manipulations. For large-scale productionof chimeric capsomeres, protein expression can be carried out usingeither viral or eukaryotic vectors. Preferable vectors include any ofthe well known prokaryotic expression vectors, recombinantbaculoviruses, COS cell specific vectors, vaccinia recombinants, oryeast-specific expression constructs. When recombinant proteins are usedto provide capsomeres of the invention, the proteins may first beisolated from the host cell of its expression and thereafter incubatedunder conditions which permit self-assembly to provide capsomeres.Alternatively, the proteins may be expressed under conditions whereincapsomeres are formed in the host cell.

[0021] The invention also contemplates processes for producingcapsomeres of the vaccine formulations. In one method, L1 proteins areexpressed from DNA encoding six additional histidines at the carboxyterminus of the L1 protein coding sequence L1 proteins expressed withadditional histidines (His L1 proteins) are most preferably expressed inE. coli and the His L1 proteins can be purified using nickel affinitychromatography. His L1 proteins in cell lysate are suspended in adenaturation buffer, for example, 6 M guanidine hydrochloride or abuffer of equivalent denaturing capacity, and then subjected to nickelchromatography. Protein eluted from the nickel chromatography step isrenatured, for example in 150 mM NaCl, 1 mM CaCl_(2,) 0.01% Triton-X100, 10 mM HEPES (N-2-hydroxymethyl piperazine-N′-2 ethane sulfonicacid), pH 7.4. According to a preferred method of the invention,assembly of capsomeres takes place after dialysis of the purifiedproteins, preferably after dialysis against 150 mM NaCl, 25 mM Ca²⁺, 10%DMSO (dimethyl sulfoxide). 0.1% Triton-X 100. 10 mM Tris[tris-(hydroxymethyl) amino-methane] acetic acid with a pH value of 5.0.

[0022] Formation of capsomeres can be monitored by electron microscopy,and, in instances wherein capsomeres are comprised of fusion proteins,the presence of various protein components in the assembled capsomerecan be confirmed by Western blot analysis using specific antisera.

[0023] According to the present invention, methods are provided fortherapeutic treatment of individuals infected with HPV comprising thestep of administering to a patient in need thereof an amount of avaccine formulation of the invention effective to reduce the level ofHPV infection. The invention also provide methods for prophylactictreatment of individuals susceptible to HPV infection comprising thestep of administering to an individual susceptible to HPV infection anamount of a vaccine formulation of the invention effective to preventHPV infection. While infected individuals can be easily identified usingstandard diagnostic techniques, susceptible individuals may beidentified, for example, as those engaged in sexual relations with aninfected individual. However, due to the high frequency of HPVinfection, all sexually active persons are susceptible to papillomavirus infection.

[0024] Administration of a vaccine formulation can include one or moreadditional components such as pharmaceutically acceptable carriers,diluents, adjuvants, and/or buffers. Vaccines may be administered at asingle time or at multiple times. Vaccine formulation of the inventionmay be delivered by various routes including, for example, oral,intravenous, intramuscular, nasal, rectal, transdermal, vaginal,subcutaneous, and intraperitoneal administration.

[0025] Vaccine formulations of the invention offer numerous advantageswhen compared to conventional vaccine preparations. As part of atherapeutic vaccination, capsomeres can promote elimination ofpersistently infected cells in, for example, patients with CIN orcervical carcinoma. Additionally, therapeutic vaccinations of this typecan also serve a prophylactic purpose in protecting patients with CINlesions from re-infection. As an additional advantage, capsomeres canescape neutralization by pre-existing anticapsid antibodies and therebyposses longer circulating half-life as compared to chimeric virus-likeparticles.

[0026] Vaccine formulations comprising chimeric capsomeres can providethe additional advantage of increased antigenicity of both proteincomponents of the fusion protein from which the capsomere is formed. Forexample, in a VLP, protein components of the underlying capsomere may beburied in the overall structure as a result of internalized positioningwithin the VLP itself. Similarly, epitopes of the protein components maybe sterically obstructed as, a result of capsomere-to-capsomere contact,and therefore unaccessible for eliciting an immune response. Preliminaryresults using L1/E7 fusion proteins to produce VLPs support thisposition in that no antibody response was detected against the E7component. This observation is consistent with previous results whichindicate that the carboxy terminal region of L1 forms inter-pentamericarm structures that allow assembly of capsomeres into capsids [Garcia,et al., J. Virol. 71: 2988-2995 (1997)]. Presumably in a chimericcapsomere structure, both protein components of the fusion proteinsubstructure are accessible to evoke an immune response. Capsomerevaccines would therefore offer the additional advantage of increasedantigenicity against any protein component, including, for example,neutralizing epitopes from other virus proteins, expressed as a fusionwith L1 amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is illustrated by the following examples.Example 1 describes construction of expression vectors to producefusion, or chimeric, viral proteins. Example 2 relates to generation ofrecombinant baculoviruses for expression of viral proteins. Example 3addresses purification of capsomeres. Example 4 describes animmunization protocol for production of antisera and monoclonalantibodies. Example 5 provides a peptide ELISA to quantitate capsomereformation. Example 6 describes an antigen capture ELISA to quantitatecapsomere formation. Example 7 provides a hemagglutinin assay to assayfor the induction of neutralizing antibodies.

EXAMPLE 1 Construction of Chimeric L1 Genes

[0028] DNA, encoding the BPV 16 L1 open reading frame was excised fromplasmid 16-114/k-L1/L2-pSynxtVI* [Kirnbauer et al., J. Virol.67:6929-6936 (1994)] using Bg/II and the resulting fragment subclonedinto pUC19 (New England Biolabs, Beverly, Mass.) previously linearizedat the unique BamHI restriction site. Two basic expression constructswere first generated to permit subsequent insertion of DNA to allowfusion protein expression. One construct encoded HPV 16 L1Δ310 having anine amino acid deletion: the deleted region was known to show low levelhomology with all other papilloma virus L1 proteins. The secondconstruct, HPV 16 L1 ΔC, encoded a protein having a 34 amino aciddeletion of the carboxy terminal L1 residues. Other constructs includean EcoRV restriction site at the position of the deletion forfacilitated insertion of DNA encoding other protein sequences. Additionof the EcoRV site encodes two non-L1 protein amino acids, aspartate andisoleucine.

[0029] A. Generation of an HPV 16 L1Δ310 Expression Construct

[0030] Two primers (SEQ ID NOs: 5 and 6) were designed to amplify thepUC19 vector and the complete HPV 16 L1 coding sequence, exceptnucleotides 916 through 942 in SEQ ID NO: 1. Primers were synthesized toalso introduce a unique EcoRV restriction site (underlined in SEQ IDNOs: 5 and 6) at the termini of the amplification product.CCCCGATATCGCCTTTAATGTATAAATCGTCTGG SEQ ID NO:5CCCCGATATCTCAAATTATTTTCCTACACCTAGTG SEQ ID NO:6

[0031] The resulting PCR product was digested with EcoRV to providecomplementary ends and the digestion product circularized by ligation.Ligated DNA was transformed into E. coli using standard techniques andplasmids from resulting colonies were screened for the presence of anEcoRV restriction site. One clone designated HPV 16 L1 Δ310 wasidentified as having the appropriate twenty-seven nucleotide deletionand this construct was used to insert DNA fragments encoding other HPV16 proteins at the EcoRV site as discussed below.

[0032] B. Generation of an HPV 16 L1 ΔC Expression Constructs

[0033] Two primers (SEQ ID NOs: 7 and 8) were designed complementary tothe HPV 16 L1 open reading frame such that the primers abutted eachother to permit amplification in reverse directions on the template DNAcomprising HPV 16 L1-encoding sequences in pUC19 described above.AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC SEQ ID NO:7AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACG SEQ ID NO:8

[0034] Each primer introduced an EcoRV restriction site at the terminusof the amplification product. In the downstream primer (SEQ ID NO: 8),the EcoRV site was followed by a TAA translational stop codon positionedsuch that the amplification product, upon ligation of the EcoRV ends tocircularize, would include deletion of the 34 carboxy terminal L1 aminoacids. PCR was performed to amplify the partial L1 open reading frameand the complete vector. The amplification product was cleaved withEcoRV, circularized with T4 DNA ligase, and transformed into E. coli DH5α cells. Plasmids from viable clones were analyzed for the presence ofan EcoRV site which would linearize the plasmid. One positive constructdesignated pUCHPV16L1ΔC was identified and used to insert DNA from otherHPV 16 proteins utilizing the EcoRV site.

[0035] C. Insertion of DNA fragments into HPV 16 L1 Δ310 and HPV16L1ΔC

[0036] DNA fragments of HPV 16 E7 encoding amino acids 1-50, 1-60, 1-98,25-75, 40-98, 50-98 in SEQ ID NO: 4 were amplified using primers thatintroduced terminal 5′ EcoRV restriction sites in order to facilitateinsertion of the fragment into either HPV 16 L1 Δ310 and HPV16L1ΔCmodified sequence. In the various amplification reactions, primer E7.1(SEQ ID NO: 9) was used in combination with primer E7.2 (SEQ ID NO: 10)to generate a DNA fragment encoding E7 amino acids 1-50: with primerE7.3 (SEQ ID NO: 11) generate a DNA fragment encoding E7 amino acids1-60: or with primer E7.4 (SEQ ID NO: 12) generate a DNA fragmentencoding E7 amino acids 1-98. In other amplification reactions, primerpairs E7.5 (SEQ ID NO: 13) and E7.6 (SEQ ID NO: 14) were used to amplifya DNA fragment encoding E7 amino acids 25-75: E7.7 (SEQ ID NO: 15) andE7.4 (SEQ ID NO: 12) were used to amplify a DNA fragment encoding E7amino acids 40-98; and E7.8 (SEQ ID NO: 16) and E7.4 (SEQ ID NO: 12)were used to amplify a DNA fragment encoding E7 amino acids 50-98.Primer E7.1 AAAAGATATCCATGCATGGAGATACACCTACATTGC SEQ ID NO:9 Primer E7.2TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC SEQ ID NO:10 Primer E7.3TTTTGATATCCTTGCAACAAAAGGTTACAATATTGTAATGGGCC SEQ ID NO:11 Primer E7.4AAAAGATATCTGGTTTCTGAGAACAGATGGGGCAC SEQ ID NO:12 Primer E7.5TTTTGATATCGATTATGAGCAATTAAATGACAGCTCAG SEQ ID NO:13 Primer E7.6TTTTGATATCGTCTACGTGTGTGCTTTGTACGCAC SEQ ID NO:14 Primer E7.7TTTATCGATATCGGTCCAGCTGGACAAGCAGAACCGGAC SEQ ID NO:15 Primer E7.8TTTTGATATCGATGCCCATTACAATATTGTAACCTTTTG SEQ ID NO:16

[0037] Similarly, nucleotides from DNA encoding the influenza matrixprotein (SEQ ID NO: 17) was amplified using the primer pair set out inSEQ ID NOs: 19 and 20. Both primers introduced an EcoRV restriction sitein the amplification product. TTTTGATATCGATATGGAATGGCTAAAGACAAGACCAATCSEQ ID NO:19 TTTTGATATCGTTGTTTGGATCCCCATTCCCATTG SEQ ID NO:20

[0038] PCR products from each amplification reaction were cleaved withEcoRV and inserted into the EcoRV site of either the HPV 16 L1 Δ310 andHPV16L1ΔC sequences previously linearized with the same enzyme. In orderto determine the orientation of inserts in plasmids encoding E7 aminoacids 25-75 and 50-98 and plasmid including influenza matrix protein,ClaI digestion was employed, taking advantage of a restriction siteoverlapping the newly created EcoRV restriction site (GATATCGAT) andincluded in the upstream primer. For the three expression constructsincluding the initiating methionine of HPV16 E7, insert orientation wasdetermined utilizing a NslI restriction site within the E7 codingregion.

[0039] Once expression constructs having appropriate inserts wereidentified, the protein coding region for both L1 and inserted aminoacids was excised as a unit using restriction enzymes XbaI and Smal andthe isolated DNA ligated into plasmid pVL1393 (Invitrogen) to generaterecombinant baculoviruses.

[0040] D. Elimination of EcoRV Restriction Sites in ExpressionConstructs

[0041] The HPV 16 L1 ΔC sequence includes DNA from the EcoRV site thatresults in translation of amino acids not normally found in wild-type L1polypeptides. Thus, a series of expression constructions was designed inwhich the artificial EcoRv site was eliminated. The L1 sequence for thisseries of expression constructs was designated HPV 16L1ΔC*.

[0042] To generate an expression construct containing the HPV 16L1ΔC*sequence, two PCR reactions were preformed to amplify two overlappingfragments from the pUC-HPV16 L1ΔC encoding E7 amino acids 1-50. Theresulting DNA fragments overlapped at the position of the L1/E7 boundarybut did not contain the two EcoRV restriction sites. Fragment 1 wasgenerated using primers P1 (SEQ ID NO: 21) and P2 (SEQ ID NO: 22) andfragment 2 using primers P3 (SEQ ID NO: 23) and P4 (SEQ ID NO: 24).Primer P1 GTTATGACATACATACATTCTATG SEQ ID NO:21 Primer P2CCATGCATTCCTGCTTGTAGTAAAAATTTGCGTCC SEQ ID NO:22 Primer P3CTACAAGCAGGAATGCATGGAGATACACC SEQ ID NO:23 Primer P4CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCTG SEQ ID NO:24

[0043] Following the first two amplification reactions, the two purifiedproducts were used as templates in another PCR reaction using primers P1and P4 only. The resulting amplification product was digested withenzymes EcoNI and HindIII inserted into the HPV 16L1 ΔC expressionconstruct described above following digestion with the same enzymes. Theresulting expression construct differed from the original HPV16L1ΔCconstruct with DNA encoding L1 and E7 amino acids 1-50 by loss of thetwo internal EcoRV restriction sites. The first EcoRV site was replacedby DNA encoding native L1 alanine and glycine amino acids in thisposition and the second was replaced by a translational stop signal. Inaddition, the expression constrict, designated HPV 16 L1ΔC* E7 1-52,contained the first 52 amino acids of HPV 16 E7 as a result of usingprimer P4 which also encodes E7 amino acids residues histidine atposition 51 and tyrosine at position 52. HPV 16 L1ΔC* E7 1-52 was thenused to generate additional HPV 16 L1 ΔC expression constructs furtherincluding DNA encoding E7 amino acids 1-55 using primer P1 (SEQ ID NO:21) in combination with primer P5 (SEQ ID NO: 25), E7 amino acids 1-60with primer pair P1 and P6 (SEQ ID NO: 26), and E7 amino acids 1-65 withprimer pair P1 and P7 (SEQ ID NO: 27). The additional aminoacid-encoding DNA sequences in the amplification products arose fromdesign of the primers to include additional nucleotides for the desiredamino acids. Primer P5 CATCTGAAGCTTAACAATATTGTAATGGGCTCTGTCCG SEQ IDNO:25 Primer P6 CATCTGAAGCTTACTTGCAACAAAAGGTTACAATATTGTAATGGGCTCTGTCCGSEQ ID NO:26 Primer P7CATCTGAAGCTTAAAGCGTAGAGTCACACTTGCAACAAAAGGTTACAATATTGTAATGGGCTCTGTCCGSEQ ID NO:27

[0044] Similarly, HPV 16 L1ΔC* E7 1-70 was venerated using template DNAencoding HPV 16 L1ΔC* E7 1-66 and the primer pair P1 and P8 (SEQ ID NO:28). Primer P8 CATCTGAAGCTTATTGTACGCACAACCGAAGCGTAGAGTCACACTTG SEQ IDNO:28

[0045] Following each PCR reaction, the amplification products weredigested with EcoNI and HindIII and inserted into HPV16L1ΔC previouslydigested with the same enzymes. Sequences of each constructs weredetermined using an Applied Biosystems Prism 377 sequencing instrumentwith fluorescent chain terminating dideoxynucleotides [Prober et al.,Science 238:336-341 (1987)].

EXAMPLE 2 Generation of Recombinant Baculoviruses

[0046]Spodoprera frugiperda (Sf9) cells were grown in suspension ormonolayer cultures at 27° in TNMFH medium (Sigma) supplemented with 10%fetal calf serum and 2 mM glutamine. For HPV 16 L1-based recombinantbaculovirus construction, Sf9 cells were transfected with 10 μg oftransfer plasmid together with 2 μg of linearized Baculo-Gold DNA(PharMingen, San Diego, Calif.) Recombinant viruses were purified byaccording to manufacturer's suggested protocol.

[0047] To test for expression of HPV 16 L1 protein, 10⁵ Sf9 cells wereinfected with baculovirus recombinant at a multiplicity of infection(m.o.i) of 5 to 10. After incubation for three to four days at 28° C.,media was removed and cells were washed with PBS. The cells were lysedin SDS sample buffer and analyzed by SDS-PAGE and Western blotting usinganti-HPV16 L1 and anti-HPV16 E7 antibodies.

[0048] In order to determine which of the chimeric L1 protein expressionconstructs would preferentially produce capsomeres, extracts fromtransfected cells were subjected to gradient centrifugation. Fractionsobtained from the gradient were analyzed for L1 protein content byWestern blotting and for VLP formation by electron microscopy. Theresults are shown in Table 1.

[0049] The intact HPV L1 protein, as well as the expression products HPV16 L1Δ310 and HPV 16 L1ΔC, each were shown to produce capsomeres andvirus-like particles in equal proportions. When E7 coding sequences wereinserted into the HPV 16 L1Δ310 vector, only fusion proteins includingE7 amino acids 1 to 50 produced gave rise to detectable capsomereformation.

[0050] When E7 encoding DNA was inserted into the HPV 16 L1ΔC vector,all fusion proteins were found to produce capsomeres; chimeric proteinsincluding E7 amino acid residues 40-98 produced the highest level ofexclusively capsomere structures. Chimeric proteins including E7 aminoacids 1-98 and 25-75 both produced predominantly capsomeres, eventhorough virus-like particle formation was also observed. The chimericprotein including E7 amino acids 1-60 resulted in nearly equal levels ofcapsomere and virus-like particle production.

[0051] When E7 sequences were inserted into the HPV 16 L1Δ*C vector, allfusion proteins were shown to produce capsomeres. Insertion of DNAencoding E7 residues 1-52, 1-55, and 1-60 produced the highest level ofcapsomeres, but equal levels of virus-like particle production wereobserved. While insertion of DNA encoding E7 DNA for residues 1-65,1-70, 25-75, 40-98, and 1-98 resulted in comparatively lower levels orundetectable levels of capsid, capsomeres were produced in highquantities. TABLE 1 Capsomeree and Capsid Forming Capacity of ChimericHPV L1 Proteins L1 Expression Capsomere Capsid Construct Insert YieldYield HVP 16 L1 None +++++ +++++ HPV 16 L1Δ310 None +++ ++ HPV 16 L1ΔCNone ++++ ++++ HPV 16 L1Δ310 E7 1-98 − − HPV 16 L1Δ310 E7 1-50 ++ − HPV16 L1Δ310 E7 25-75 − − HPV 16 L1Δ310 E7 50-98 − − HPV 16 L1ΔC E7 1-98+++ + HPV 16 L1ΔC E7 25-75 +++ + HPV 16 L1ΔC E7 50-98 + + HPV 16 L1ΔC E71-60 +++++ +++++ HPV 16 L1ΔC E7 40-98 ++++ − HPV 16 L1ΔC Influenza +++ +HPV 16 L1Δ*C E7 1-52 +++++ +++++ HPV 16 L1Δ*C E7 1-55 +++++ +++++ HPV I6L1Δ*C E7 1-60 +++ ++++ HPV 16 L1Δ*C E7 1-65 ++ − HPV 16 L1Δ*C E7 1-70 ++−

EXAMPLE 3 Purification of Capsomeres

[0052] Trichopulsia ni (TN) High Five cells were grown to a density ofapproximately 2×10⁶ cells/mi in Ex-Cell 405 serum-free medium (JRHBiosciences). Approximately 2×10⁸ cells were pelleted by centrifugationat 1000×g for 15 minutes, resuspended in 20 ml of medium, and infectedwith recombinant baculoviruses at m.o.i of 2 to 5 for 1 hour at roomtemperature. After addition of 200 ml medium, cells were plated andincubated for 3 to 4 days at 27° C. Following incubation, cells wereharvested, pelleted, and resuspended in 10 ml of extraction buffer.

[0053] The following steps were performed at 4° C. Cells were sonicatedfor 45 seconds at 60 watts and the resulting cell lysate was centrifugedat 10,000 rpm in a Sorval SS34 rotor. The supernatant was removed andretained while the resulting pellet was resuspended in 6 ml ofextraction buffer, sonicated for an additional 3 seconds at 60 watts,and centrifuged again. The two supernatants were combined, layered ontoa two-step gradient containing 14 ml of 40% sucrose on top of 8 ml ofCsCl solution (4.6 g CsCl per 8 ml in extraction buffer), andcentrifuged in a Sorval AH629 swinging bucket rotor for 2 hours at27,000 rpm at 10° C. The interface region between the CsCl and thesucrose along with the CsCl complete layer were collected into 13.4 mlQuickseal tubes (Beckman) and extraction buffer added to adjust thevolume 13.4 ml. Samples were centrifuged overnight at 50,000 rpm at 20°C. in a Beckman 70 TI rotor. Gradients were fractionated (1 ml perfraction) by puncturing tubes on top and bottom with a 21-gauge needle.Fractions were collected from each tube and 2.5 μl of each fraction wereanalyzed by a 10% SDS-polyacrylamide gel and Western blotting using ananti-HPV16 L1 antibody.

[0054] Virus-like particles and capsomeres were separated from thefractions identified above by sedimentation on 10 to 50% sucrosegradients. Peak fractions from CsCl gradients were pooled and dialyzedfor 2 hours against 5 mM HEPES (pH 7.5). Half of the dialysate was usedto produce capsomeres by disassembly of intact VLPs overnight by addingEDTA (final concentration 50 mM), EGTA (50 mM), DTT (30 mM), NaCl (100mM), and Tris/HCl, pH 8.0, (10 mM). As control, NaCl and Tis/HCl onlywere added to the other half.

[0055] For analysis of capsomeres produced from disassembled VLPs, EDTA,EGTA, and DTT (final concentration 5 mM each) were added to the sucrosecushions which were centrifuged at 250,000×g for 2 to 4 hours at 4° C.Fractions were collected by puncturing tubes from the bottom. A 1:10dilution of each fraction was then analyzed by antigen capture ELISA.

EXAMPLE 4 Immunization Protocol for Production of Polyclonal Antiseraand Monoclonal Antibodies

[0056] Balb/c mice are immunized subcutaneously three times, every fourweeks with approximately 60 μg of HPV chimeric capsomeres mixed 1:1 withcomplete or incomplete Freund's Adjuvants in a total volume of 100 μl.Six weeks after the third immunization, mice are sacrificed and blood iscollected by cardiac puncture.

EXAMPLE 5 Peptide ELISA to Quantitate Capsomere Formation

[0057] Microtiter plates (Dynatech) are coated overnight with 50 μl ofpeptide E701 [Muller et al., 1982] at a concentration of 10 μg/ml inPBS. Wells are blocked for 2 hour at 37° C. with 100 μl of buffercontaining 5% BSA and 0.05% Tween 20 in PBS and washed three times withPBS containing 0.05% Tween 20. After the third wash, 50 μl of seradiluted 1:5000 in BSA/Tween 20/PBS is added to each well and incubationcarried out for 1 hour. Plates are washed again as before and 50 μl ofgoat-anti-mouse peroxidase conjugate is added at a 1:5000 dilution.After 1 hour, plates are washed and stained using ABTS substrate (0.2mg/ml, 2,2′-Azino-bis(3-ethylbenzhiazoline-β-sulfonic acid in 0.1 MNa-Acetate-Phosphate buffer (pH 4.2) with 4 μl 30% H₂O₂ per 10 ml).Extinction is measured after 1 hour at 490 nm in a Dynatech automatedplate reader.

EXAMPLE 6 Antigen Capture ELISA to Quantitate Capsomere Formation

[0058] To allow relative quantification of virus-like particles andcapsomeres in fractions of CsCl gradients, an antigen capture ELISA wasutilized. Microtiter plates were coated overnight with 50 μl/well of a1:500 dilution (final concentration of 2 μg per ml, in PBS) with aprotein A purified mouse monoclonal antibody immunospecific for HPV 16L1 (antibodies 25/C, MM07 and Ritti 1 were obtained from mice immunizedwith HPV 16 VLPs). Plates were blocked with 5% milk/PBS for 1 hour and50 μl of fractions of CsCl gradients were added for 1 hour at 37° C.using a 1:300 dilution (in 5% milk/PBS). After three washings withPBS/0.05% Tween 20, 50 μl of a polyclonal rabbit antiserum (1:3000dilution in milk/PBS), raised against HPV 16 VLPs was added and plateswere incubated at 375 for 1 hour. Plates were washed again and furtherincubated with 50 μl of a goat-anti-rabbit peroxidase conjugate (Sigma)diluted 1:5000 in PBS containing 5% milk for 1 hour. After finalwashing, plates were stained with ABTS substrate for 30 minutes andextinction measured at 490 nm in a Dynatech automated plate reader. As anegative control, the assay also included wells coated only with PBS.

[0059] To test monoclonal antibodies for capsomere specificity, VLPswith EDTA/DTT to disassemble particles. Treated particle preparationswere assayed in the antigen-capture ELISA and readings compared tountreated controls. For disassembly, 40 μl of VLPs was incubatedovernight at 4° C. in 500 μl of disruption buffer containing 30 mM DTT.50 mM EGTA, 60 mM EDTA, 100 mM NaCl, and 100 mM Tris/HCl, pH 8.0.Aliquots of treated and untreated particles were used in the abovecapture ELISA in a 1:20-1:40 dilution.

EXAMPLE 7 Hemagglutinin Inhibition Assay

[0060] In order to determine the extent to which chimeric capsomerevaccines evoke production of neutralizing antibodies, a hemagglutinationinhibition assay is carried out as briefly described below. This assayis based on previous observations that virus-like particles are capableof hemagglutinizing red blood cells.

[0061] Mice are immunized with any of a chimeric capsomere vaccine andsera is collected as described above in Example 4. As positive controls,BPV16 L1 virus like particles (VLPs) and bovine PV1 (BPV) L1 VLPs areassayed in parallel with a chimeric capsomere preparation. To establisha positive baseline, the HPV 16 or BPV1 VLPs are first incubated with orwithout sera collected from immunized mice after which red blood cellsare added. The extent to which preincubation with mouse cera inhibitsred blood cell hemagglutinization is an indication of the neutralizingcapacity of the mouse sera. The experiments are then repeated usingchimeric capsomeres in order to determine the neutralizing effect of themouse sera on the vaccine. A brief protocol for the hemagglutinationinhibition assay is described below.

[0062] One hundred microliters of heparin (1000 usp units/ml) are addedto 1 ml fresh mouse blood. Red blood cells are washed three times withPBS followed by centrifugation and resuspension in a volume of 10 ml.Next, erythrocytes are resuspended in 0.5 ml PBS and stored at 4° C. forup to three days. For the hemagglutinin assay. 70 μl of the suspensionis used per well on a 96-well plate.

[0063] Chimeric capsomere aliquots from CsCl gradients are dialyzed forone hour against 10 mM Hepes (pH 7.5) and 100 μl of two-fold serialdilutions in PBS are added to mouse erythrocytes in round-bottom 96-wellmicrotiter plates which are further incubated for 3-16 hours at 4° C.For hemagglutination inhibition, capsomeres are incubated with dilutionsof antibodies in PBS for 60 minutes at room temperature and then addedto the erythrocytes. The level of erythrocyte hemagglutination, andtherefore the presence of neutralizing antibodies, is determined bystandard methods.

[0064] In preliminary results, mouse sera generated against chimericcapsomeres comprising HPV16L1ΔC protein in association with E7 aminoacid residues 1-98 was observed to inhibit hemagglutination by HPV16VLPs, but not by BPV VLPs. The mouse sera was therefore positive forneutralizing antibodies against the human VLPs and this differentialneutralization was most likely the result of antibody specificity forepitopes against which the antibodies were raised.

[0065] Numerous modifications and variations in the invention as setforth in the above illustrative examples are expected to occur to thoseskilled in the art Consequently only such limitations as appear in theappended claims should be placed on the invention.

1 28 1518 base pairs nucleic acid single linear DNA (genomic) CDS1..1515 1 ATG TCT CTT TGG CTG CCT AGT GAG GCC ACT GTC TAC TTG CCT CCTGTC 48 Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 15 10 15 CCA GTA TCT AAG GTT GTA AGC ACG GAT GAA TAT GTT GCA CGC ACA AAC96 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 2530 ATA TAT TAT CAT GCA GGA ACA TCC AGA CTA CTT GCA GTT GGA CAT CCC 144Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45TAT TTT CCT ATT AAA AAA CCT AAC AAT AAC AAA ATA TTA GTT CCT AAA 192 TyrPhe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys 50 55 60 GTATCA GGA TTA CAA TAC AGG GTA TTT AGA ATA CAT TTA CCT GAC CCC 240 Val SerGly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro 65 70 75 80 AATAAG TTT GGT TTT CCT GAC ACC TCA TTT TAT AAT CCA GAT ACA CAG 288 Asn LysPhe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln 85 90 95 CGG CTGGTT TGG GCC TGT GTA GGT GTT GAG GTA GGT CGT GGT CAG CCA 336 Arg Leu ValTrp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro 100 105 110 TTA GGTGTG GGC ATT AGT GGC CAT CCT TTA TTA AAT AAA TTG GAT GAC 384 Leu Gly ValGly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125 ACA GAAAAT GCT AGT GCT TAT GCA GCA AAT GCA GGT GTG GAT AAT AGA 432 Thr Glu AsnAla Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140 GAA TGTATA TCT ATG GAT TAC AAA CAA ACA CAA TTG TGT TTA ATT GGT 480 Glu Cys IleSer Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly 145 150 155 160 TGCAAA CCA CCT ATA GGG GAA CAC TGG GGC AAA GGA TCC CCA TGT ACC 528 Cys LysPro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175 AATGTT GCA GTA AAT CCA GGT GAT TGT CCA CCA TTA GAG TTA ATA AAC 576 Asn ValAla Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn 180 185 190 ACAGTT ATT CAG GAT GGT GAT ATG GTT GAT ACT GGC TTT GGT GCT ATG 624 Thr ValIle Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 195 200 205 GACTTT ACT ACA TTA CAG GCT AAC AAA AGT GAA GTT CCA CTG GAT ATT 672 Asp PheThr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile 210 215 220 TGTACA TCT ATT TGC AAA TAT CCA GAT TAT ATT AAA ATG GTG TCA GAA 720 Cys ThrSer Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240CCA TAT GGC GAC AGC TTA TTT TTT TAT TTA CGA AGG GAA CAA ATG TTT 768 ProTyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe 245 250 255GTT AGA CAT TTA TTT AAT AGG GCT GGT GCT GTT GGT GAA AAT GTA CCA 816 ValArg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260 265 270GAC GAT TTA TAC ATT AAA GGC TCT GGG TCT ACT GCA AAT TTA GCC AGT 864 AspAsp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285TCA AAT TAT TTT CCT ACA CCT AGT GGT TCT ATG GTT ACC TCT GAT GCC 912 SerAsn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300CAA ATA TTC AAT AAA CCT TAT TGG TTA CAA CGA GCA CAG GGC CAC AAT 960 GlnIle Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn 305 310 315320 AAT GGC ATT TGT TGG GGT AAC CAA CTA TTT GTT ACT GTT GTT GAT ACT 1008Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr 325 330335 ACA CGC AGT ACA AAT ATG TCA TTA TGT GCT GCC ATA TCT ACT TCA GAA 1056Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu 340 345350 ACT ACA TAT AAA AAT ACT AAC TTT AAG GAG TAC CTA CGA CAT GGG GAG 1104Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360365 GAA TAT GAT TTA CAG TTT ATT TTT CAA CTG TGC AAA ATA ACC TTA ACT 1152Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr 370 375380 GCA GAC GTT ATG ACA TAC ATA CAT TCT ATG AAT TCC ACT ATT TTG GAG 1200Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu 385 390395 400 GAC TGG AAT TTT GGT CTA CAA CCT CCC CCA GGA GGC ACA CTA GAA GAT1248 Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp 405410 415 ACT TAT AGG TTT GTA ACC TCC CAG GCA ATT GCT TGT CAA AAA CAT ACA1296 Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr 420425 430 CCT CCA GCA CCT AAA GAA GAT CCC CTT AAA AAA TAC ACT TTT TGG GAA1344 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435440 445 GTA AAT TTA AAG GAA AAG TTT TCT GCA GAC CTA GAT CAG TTT CCT TTA1392 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu 450455 460 GGA CGC AAA TTT TTA CTA CAA GCA GGA TTG AAG GCC AAA CCA AAA TTT1440 Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe 465470 475 480 ACA TTA GGA AAA CGA AAA GCT ACA CCC ACC ACC TCA TCT ACC TCTACA 1488 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr485 490 495 ACT GCT AAA CGC AAA AAA CGT AAG CTG TAA 1518 Thr Ala Lys ArgLys Lys Arg Lys Leu 500 505 505 amino acids amino acid linear protein 2Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 1015 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 2530 Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 4045 Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys 50 5560 Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro 65 7075 80 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln 8590 95 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro100 105 110 Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu AspAsp 115 120 125 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val AspAsn Arg 130 135 140 Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu CysLeu Ile Gly 145 150 155 160 Cys Lys Pro Pro Ile Gly Glu His Trp Gly LysGly Ser Pro Cys Thr 165 170 175 Asn Val Ala Val Asn Pro Gly Asp Cys ProPro Leu Glu Leu Ile Asn 180 185 190 Thr Val Ile Gln Asp Gly Asp Met ValAsp Thr Gly Phe Gly Ala Met 195 200 205 Asp Phe Thr Thr Leu Gln Ala AsnLys Ser Glu Val Pro Leu Asp Ile 210 215 220 Cys Thr Ser Ile Cys Lys TyrPro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240 Pro Tyr Gly Asp SerLeu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe 245 250 255 Val Arg His LeuPhe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260 265 270 Asp Asp LeuTyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285 Ser AsnTyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300 GlnIle Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn 305 310 315320 Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr 325330 335 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu340 345 350 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His GlyGlu 355 360 365 Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile ThrLeu Thr 370 375 380 Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser ThrIle Leu Glu 385 390 395 400 Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro GlyGly Thr Leu Glu Asp 405 410 415 Thr Tyr Arg Phe Val Thr Ser Gln Ala IleAla Cys Gln Lys His Thr 420 425 430 Pro Pro Ala Pro Lys Glu Asp Pro LeuLys Lys Tyr Thr Phe Trp Glu 435 440 445 Val Asn Leu Lys Glu Lys Phe SerAla Asp Leu Asp Gln Phe Pro Leu 450 455 460 Gly Arg Lys Phe Leu Leu GlnAla Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480 Thr Leu Gly Lys ArgLys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495 Thr Ala Lys ArgLys Lys Arg Lys Leu 500 505 297 base pairs nucleic acid single linearDNA (genomic) CDS 1..294 3 ATG CAT GGA GAT ACA CCT ACA TTG CAT GAA TATATG TTA GAT TTG CAA 48 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr MetLeu Asp Leu Gln 1 5 10 15 CCA GAG ACA ACT GAT CTC TAC TGT TAT GAG CAATTA AAT GAC AGC TCA 96 Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln LeuAsn Asp Ser Ser 20 25 30 GAG GAG GAG GAT GAA ATA GAT GGT CCA GCT GGA CAAGCA GAA CCG GAC 144 Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln AlaGlu Pro Asp 35 40 45 AGA GCC CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG TGTGAC TCT ACG 192 Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys AspSer Thr 50 55 60 CTT CGG TTG TGC GTA CAA AGC ACA CAC GTA GAC ATT CGT ACTTTG GAA 240 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr LeuGlu 65 70 75 80 GAC CTG TTA ATG GGC ACA CTA GGA ATT GTG TGC CCC ATC TGTTCT CAG 288 Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys SerGln 85 90 95 AAA CCA TAA 297 Lys Pro 98 amino acids amino acid linearprotein 4 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp LeuGln 1 5 10 15 Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn AspSer Ser 20 25 30 Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala GluPro Asp 35 40 45 Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys AspSer Thr 50 55 60 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg ThrLeu Glu 65 70 75 80 Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro IleCys Ser Gln 85 90 95 Lys Pro 34 base pairs nucleic acid single linearother nucleic acid /desc = “Primer” 5 CCCCGATATC GCCTTTAATG TATAAATCGTCTGG 34 35 base pairs nucleic acid single linear other nucleic acid/desc = “Primer” 6 CCCCGATATC TCAAATTATT TTCCTACACC TAGTG 35 40 basepairs nucleic acid single linear other nucleic acid /desc = “Primer” 7AAAGATATCT TGTAGTAAAA ATTTGCGTCC TAAAGGAAAC 40 44 base pairs nucleicacid single linear other nucleic acid /desc = “Primer” 8 AAAGATATCTAATCTACCTC TACAACTGCT AAACGCAAAA AACG 44 35 base pairs nucleic acidsingle linear other nucleic acid /desc = “Primer” 9 AAAAGATATCATGCATGGAG ATACACCTAC ATTGC 35 34 base pairs nucleic acid single linearother nucleic acid /desc = “Primer” 10 TTTTGATATC GGCTCTGTCC GGTTCTGCTTGTCC 34 44 base pairs nucleic acid single linear other nucleic acid/desc = “Primer” 11 TTTTGATATC CTTGCAACAA AAGGTTACAA TATTGTAATG GGCC 4435 base pairs nucleic acid single linear other nucleic acid /desc =“Primer” 12 AAAAGATATC TGGTTTCTGA GAACAGATGG GGCAC 35 38 base pairsnucleic acid single linear other nucleic acid /desc = “Primer” 13TTTTGATATC GATTATGAGC AATTAAATGA CAGCTCAG 38 35 base pairs nucleic acidsingle linear other nucleic acid /desc = “Primer” 14 TTTTGATATCGTCTACGTGT GTGCTTTGTA CGCAC 35 39 base pairs nucleic acid single linearother nucleic acid /desc = “Primer” 15 TTTATCGATA TCGGTCCAGC TGGACAAGCAGAACCGGAC 39 39 base pairs nucleic acid single linear other nucleic acid/desc = “Primer” 16 TTTTGATATC GATGCCCATT ACAATATTGT AACCTTTTG 39 294base pairs nucleic acid single linear DNA (genomic) CDS 1..291 17 ATGAGT CTT CTA ACC GAG GTC GAA ACG CTT ACC AGA AAC GGA TGG GAG 48 Met SerLeu Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Glu 1 5 10 15 TGCAAA TGC AGC GAT TCA AGT GAT CCT CTC ATT ATC GCA GCG AGT ATC 96 Cys LysCys Ser Asp Ser Ser Asp Pro Leu Ile Ile Ala Ala Ser Ile 20 25 30 ATT GGGATC TTG CAC TTG ATA TTG TGG ATT TTT TAT CGT CTT TTC TTC 144 Ile Gly IleLeu His Leu Ile Leu Trp Ile Phe Tyr Arg Leu Phe Phe 35 40 45 AAA TGC ATTTAT CGT CGC CTT AAA TAC GGT TTG AAA AGA GGG CCT TCT 192 Lys Cys Ile TyrArg Arg Leu Lys Tyr Gly Leu Lys Arg Gly Pro Ser 50 55 60 ACG GAA GGA GCGCCT GAG TCT ATG AGG GAA GAA TAT CGG CAG GAA CAG 240 Thr Glu Gly Ala ProGlu Ser Met Arg Glu Glu Tyr Arg Gln Glu Gln 65 70 75 80 CAG AGT GCT GTGGAT GTT GAC GAT GTT CAT TTT GTC AAC ATA GAG CTG 288 Gln Ser Ala Val AspVal Asp Asp Val His Phe Val Asn Ile Glu Leu 85 90 95 GAG TAA 294 Glu 97amino acids amino acid linear protein 18 Met Ser Leu Leu Thr Glu Val GluThr Leu Thr Arg Asn Gly Trp Glu 1 5 10 15 Cys Lys Cys Ser Asp Ser SerAsp Pro Leu Ile Ile Ala Ala Ser Ile 20 25 30 Ile Gly Ile Leu His Leu IleLeu Trp Ile Phe Tyr Arg Leu Phe Phe 35 40 45 Lys Cys Ile Tyr Arg Arg LeuLys Tyr Gly Leu Lys Arg Gly Pro Ser 50 55 60 Thr Glu Gly Ala Pro Glu SerMet Arg Glu Glu Tyr Arg Gln Glu Gln 65 70 75 80 Gln Ser Ala Val Asp ValAsp Asp Val His Phe Val Asn Ile Glu Leu 85 90 95 Glu 40 base pairsnucleic acid single linear other nucleic acid /desc = “Primer” 19TTTTGATATC GATATGGAAT GGCTAAAGAC AAGACCAATC 40 35 base pairs nucleicacid single linear other nucleic acid /desc = “Primer” 20 TTTTGATATCGTTGTTTGGA TCCCCATTCC CATTG 35 24 base pairs nucleic acid single linearother nucleic acid /desc = “Primer” 21 GTTATGACAT ACATACATTC TATG 24 35base pairs nucleic acid single linear other nucleic acid /desc =“Primer” 22 CCATGCATTC CTGCTTGTAG TAAAAATTTG CGTCC 35 29 base pairsnucleic acid single linear other nucleic acid /desc = “Primer” 23CTACAAGCAG GAATGCATGG AGATACACC 29 36 base pairs nucleic acid singlelinear other nucleic acid /desc = “Primer” 24 CATCTGAAGC TTAGTAATGGGCTCTGTCCG GTTCTG 36 38 base pairs nucleic acid single linear othernucleic acid /desc = “Primer” 25 CATCTGAAGC TTATCAATAT TGTAATGGGCTCTGTCCG 38 54 base pairs nucleic acid single linear other nucleic acid/desc = “Primer” 26 CATCTGAAGC TTACTTGCAA CAAAAGGTTA CAATATTGTAATGGGCTCTG TCCG 54 69 base pairs nucleic acid single linear othernucleic acid /desc = “Primer” 27 CATCTGAAGC TTAAAGCGTA GAGTCACACTTGCAACAAAA GGTTACAATA TTGTAATGGG 60 CTCTGTCCG 69 47 base pairs nucleicacid single linear other nucleic acid /desc = “Primer” 28 CATCTGAAGCTTATTGTACG CACAACCGAA GCGTAGAGTC ACACTTG 47

What is claimed is:
 1. A fusion protein comprising: an amino acidsequence from a first papilloma-virus specific (PVS) protein; and anamino acid sequence from a second PVS protein, wherein said fusionprotein comprises only PVS protein amino acid sequences.
 2. A fusionprotein in accordance with claim 1, comprising amino acid sequences frommore than two PVS proteins.
 3. A fusion protein in accordance with claim1, wherein at least one of the amino acid sequences from a PVS proteincomprises at least one deletion from the naturally-occurring PVS proteinsequence.
 4. A fusion protein in accordance with claim 1, wherein saidfirst PVS protein is a L protein or B protein.
 5. A fusion protein inaccordance with claim 4, wherein the L protein is L1.
 6. A fusionprotein in accordance with claim 1, wherein the E protein is E7 protein.7. A fusion protein in accordance with claim 1, wherein the first PVSprotein is a L protein, and the second PVS protein is an B protein.
 8. Afusion protein in accordance with claim 7, wherein said L protein is L1and said B protein is E7.
 9. A fusion protein in accordance with claim8, wherein the PVS proteins are human PVS proteins.
 10. A fusion proteinin accordance with claim 9, wherein said amino acid sequence from saidL1 protein does not contain the 34 amino acid sequence of the carboxyterminus.
 11. A nucleic acid sequence encoding the fusion protein ofclaim
 1. 12. A nucleic acid sequence in accordance with claim 11 that isa DNA.
 13. A therapeutic composition comprising the fusion protein ofclaim
 1. 14. A prophylactic vaccine formulation comprising the fusionprotein of claim
 1. 15. A method of making the fusion protein of claim1, comprising recombinant expression of a nucleic acid sequence encodingsaid fusion protein, and substantially isolating said fusion protein.16. A therapeutic method of treating an animal having a papilloma virusinfection, comprising administration of a pharmaceutical compositioncomprising the fusion protein of claim 1 in an amount and for a periodof time sufficient to reduce the level of papilloma virus infection. 17.A therapeutic method in accordance with claim 16, wherein the animal isa human and the PVS proteins are human PVS proteins.
 18. A prophylacticmethod of inhibiting a papilloma virus infection in an animal,comprising administration of a pharmaceutical composition comprising thefusion protein of claim 1 in an amount and for a period of timesufficient to effectively inhibit papilloma virus infection.
 19. Atherapeutic method in accordance with claim 18, wherein the animal is ahuman and the PVS proteins are human PVS proteins.